This research aims to better understand the role of cell signaling in trigeminal sensory nerve placode development. Specifically, Dr. Stark will utilize gene misexpression techniques to determine the sufficiency and necessity of Frizzled-7 and FREK in the specification of trigeminal placode cells within the cranial ectoderm, and the subsequent delamination of those cells from the epithelial layer. While several genes are known to be expressed in cranial placodes, the molecules identified for investigation in this study are dynamically expressed at key stages of trigeminal placode cell specification and delamination. In addition, these genes likely play a role in embryonic cell signaling, since both genes encode receptor molecules; Frizzled-7 encodes a Wnt receptor and FREK encodes an FGF receptor. Cell signaling through these and other receptor families is a fundamental process in biology, especially during embryonic development. The results obtained from this research will therefore have a broad impact on the biological sciences, and will be especially useful to developmental and cancer biologists. The methods and experiments outlined in this application are based on extensive research by others showing that functional and inhibitory receptor constructs can serve to elucidate receptor function. Dr. Stark and his colleagues have preliminarily constructed functional and inhibitory expression constructs for both Frizzled-7 and FREK. They will utilize these constructs in the chick embryo model system to fulfill two specific objectives: 1) Analyze the role of Frizzled-7 early trigeminal placode formation, and 2) Analyze the role of FREK in the delamination of trigeminal placode cells. Proven techniques of gene misexpression will be utilized in this study, including RCAS retroviral mediated gene delivery and in ovo electroporation. For both specific aims, inhibitory gene construct expression will act to block normal cell signaling through the given receptor, while ectopic expression of functional gene constructs will expand the domain of cells receptive to the proper signal. By analyzing downstream markers of the trigeminal placode and ganglion, it will be possible to determine the effect of gene misexpression. This research will therefore address the functional significance of Frizzled-7 and FREK in early trigeminal placode development.